bioRxiv preprint doi: https://doi.org/10.1101/2020.01.28.922179; this version posted January 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Integrated Analysis of Methylome and Transcriptome Following Developmental Atrazine Exposure in Zebrafish Reveals Aberrant Gene-Specific Methylation of Neuroendocrine and Reproductive Pathways Chris Bryan1, Li Lin2, Junkai Xie2, Janiel Ahkin Chin Tai3, Katharine A. Horzmann3,a, Kyle Wettschurack2, Min Zhang1, Jennifer Freeman3,4, Chongli Yuan2,4* 1 Department of Statistics, Purdue University, West Lafayette, IN, 47906, U.S.A. 2 Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN, 47906, U.S.A. 3 School of Health Science, Purdue University, West Lafayette, IN, 47906, U.S.A. 4 Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN, 47906, U.S.A. aCurrent affiliation: Department of Pathobiology, Auburn University College of Veterinary Medicine, Auburn, AL * To whom correspondence should be addressed. Tel:+ 1 765 494 5824; Fax: + 1 765 494 0805; Email: [email protected] Present Address: Chongli Yuan, Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN, 47906, U.S.A. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.28.922179; this version posted January 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. ABSTRACT Atrazine (ATZ) is one of the most commonly used herbicides in the United States. Previous studies have hypothesized the role of ATZ as an endocrine disruptor (EDC), and developmental exposure to ATZ has been shown to lead to behavioral and morphological alterations. Specific epigenetic mechanisms responsible for these alterations, however, are yet to be elucidated. In this study, we exposed zebrafish embryos to 0.3, 3, and 30 ppb (µg/L) of ATZ for 72 hours post fertilization. We performed whole-genome bisulfite sequencing (WGBS) to assess the effects of developmental ATZ exposure on DNA methylation in female fish brains. The number of differentially methylated genes (DMG) increase with increasing dose of treatments. DMGs are enriched in neurological pathways with extensive methylation changes consistently observed in neuroendocrine and reproductive pathways. To assess the effects of DNA methylation on gene expression, we integrated our data with transcriptomic data. Four genes, namely CHD9, FRAS1, PID1, and PCLO, were differentially expressed and methylated in each dose. Overall, this study identifies specific genes and pathways with aberrant methylation and expression following ATZ exposure as targets to elucidate the molecular mechanisms of ATZ toxicity and presents ATZ-induced site-specific DNA methylation as a potential mechanism driving aberrant gene expression. bioRxiv preprint doi: https://doi.org/10.1101/2020.01.28.922179; this version posted January 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. INTRODUCTION Atrazine (6-chloro-N-ehtyl-N-(1-methylethyl)-1,3,5-triazine-2,4-diamine. ATZ) is a widely used agriculture herbicide that controls the growth of broadleaf and weeds. The estimated total annual usage of atrazine is ~ 76.5 million pounds in the U.S., making it the second most commonly used herbicide in the country(1). ATZ has a relatively high water solubility(2) , and thus can be easily traced in surface and ground water by rainfalls and tile drainages(3–5). ATZ intake from contaminated water supplies has a wide range of adverse health outcomes, including disruption of the hypothalamic-related axis in the brain by inhibiting the luteinizing hormone production and decreasing testosterone production in rats and humans and is thus known to be a potential endocrine disrupter(6–8). Studies have also shown that ATZ exposure can demasculinize and feminize males while disrupting ovarian function in female rats(9). Recent studies also suggest that ATZ disrupts endocrine function by altering the hypothalamic-pituitary-gonadal (HPG) axis(10). For example, a recent work utilizing a female zebrafish animal model has found that ATZ exposure can disrupt the production of luteinizing hormone, follicle stimulating hormone and gonadotropin-releasing hormone, all of which are essential for female reproductive function(11). In addition to its endocrine disruption functions, ATZ exposure has also been associated with several neurological disorders. Studies using a zebrafish animal model have found that ATZ exposure can introduce significant alterations to neurotransmission pathways resulting in significant decreases of serotonin metabolites in female fish(12). Similar observations were made in mouse models, but ATZ exposure was found to primarily impact dopaminergic neurons(13– 16). Interestingly, a recent epidemiological study shows that agricultural workers are at high risk of developing depression and anxiety symptoms compared to other occupational groups(17), and the production of serotonin are associated with depression and anxiety in humans(18). Increasing evidence indicates that ATZ-induced neurotoxicity is sex-specific, with females having a higher chance of developing non-motor symptoms(19). Few studies, however, exist using female animal models, and the mechanisms of sex-specific neurotoxicity remain unclear. The toxic effects of ATZ can persist after removal of exposure source and can be inherited trans-generationally(20), suggesting the existence of underlying molecular “memory” and thus alluding to the involvement of an epigenetic mechanism. Epigenetics, including DNA methylation, histone post-translational modifications, and RNA-assisted regulation(21), refer to reversible and inheritable changes in gene expression without altering the underlying DNA sequence. Among these, the alteration of DNA and histone methylation profiles has been the most well-studied due to its abundance and relative stability compared to other types of epigenetic modifications(22–24). Specifically, a mouse bioRxiv preprint doi: https://doi.org/10.1101/2020.01.28.922179; this version posted January 28, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. study has found that ATZ exposure can result in deregulation of tissue-specific RNA transcription up to the third generation by inheritable decreases in H3K4me3, a transcription activation marker(25). Similar observations were also made in a rat model(20, 26). Our recent work has demonstrated that ATZ exposure in zebrafish can decrease global DNA methylation levels by inhibiting the activity of maintenance DNMTs and the expression level of DNMT4 and DNMT5, a zebrafish ortholog for DNMT3b(27), but that global methylation in the adult female zebrafish brain was not changed (manuscript in review). There is, however, no genome-wide studies revealing the impact of ATZ exposure on the epigenome in the brain. In this study, we used zebrafish as an animal model and performed whole-genome bisulfite sequencing using brains harvested from 9 month-old fish that were exposed to ATZ only during embryogenesis (1-72 hour post fertilization; hpf) to reveal permanent DNA methylome changes arising from embryonic ATZ exposure. Our results suggest that genes involved in key pathways related to observed behavioral changes resulting from low-dose ATZ, such as neuroendocrine function, are differentially methylated in the brain of zebrafish exposed to ATZ. After integrating the methylation data with transcriptomic data, we suggest the ATZ-induced site-specific DNA methylation as a potential mechanism driving the aberrant expression of neurological genes. MATERIAL AND METHODS Zebrafish husbandry and treatment Aliquots of a 10 parts per million (ppm; mg/L) stock solution of technical grade (98.1% purity) ATZ (CAS 1912-24-9; Chem Service, West Chester, PA) were diluted with filtered aquaria water to make exposures of 0.3, 3, and 30 ppb ATZ as previously described, with filtered aquaria water serving as the 0 ppb control(12, 28). Embryos were collected from a breeding colony of AB wild-type zebrafish (Danio rerio) maintained in a Z-Mod System (Aquatic Habitats, Apopka, FL) with a 14:10 light-dark cycle, temperature of 26-28°C, pH 7.0-7.3, and conductivity 470-550 µS. Fish and aquaria were monitored twice daily and fed a mixture of brine shrimp (Artemia franciscana; Artemia International LLC., Fairview, Texas), Zeigler adult zebrafish food (Zeigler Bros Inc., Gardners, PA), and Golden Pearls 500-800 µm (Artemia International LLC., Fairview, Texas). To obtain embryos, adult zebrafish were bred in spawning tanks according to established protocols(29, 30). Embryos were collected at the 4-8 cell stage, randomly sorted into the 0, 0.3, 3, or 30 ppb ATZ exposure groups, and incubated in petri dishes with 20 mL of exposure solution at 28.5°C. At 72 hpf, the larvae were rinsed in filtered aquaria water to end